All Flashcards
(129 cards)
1
Q
DNA Double Helix
A
- Two Polynucleotide Chains
- Antiparallel Orientation
- Sugar-Phosphate Backbone
2
Q
Bond between Base Pairs
A
Hydrogen bonds
3
Q
Bond between Sugar and Base
A
Covalent bond
4
Q
DNA replication is
A
Semi-conservative
5
Q
Meselson–Stahl Experiment (1958)
A
• Parental DNA labeled with heavy isotope
• Formation of the Hybrid DNA duplex in Generation 1
confers Semiconservative DNA Replication
6
Q
Head dsDNA to Tm
A
Denaturation
7
Q
Polymerase Chain Reaction (PCR) uses
A
Denaturation and renaturation property of the DNA used for experimentation in molecular biology
8
Q
Renaturation occurs when
A
DNA temperature is reduced to annealing temperature
Annealing = DNA to DNA or RNA to RNA
9
Q
Point Mutation
A
Loss/gain of a single base pair
Transition (purine to purine or pyrimidine to pyrimidine) or transversion (purine to pyridimidine or pyridimidie to purine)
10
Q
Insertion
A
Addition of multiple base pairs
11
Q
Purine
A
A and G
12
Q
Pyrimidine
A
C and T
13
Q
Point Mutation reversed by
A
-True reversion or second-site reversion
14
Q
Insertion mutation reversed by
A
Deletion
15
Q
Deletion mutation is
A
Irreversible
16
Q
Null mutation
A
Tests whether a gene is essential
Completely eliminates gene function
17
Q
Silent mutation
A
Mutations that do not affect protein sequence or function
18
Q
Centromere
A
“Center” of the chromosome
Highly repetitive DNA called α-satellite DNA
A-T rich
19
Q
Specific proteins that bind and/or maintain the centromere
A
- Nucleosome = incorporation of specific histone (CENP-A)
2. Chromatin-remodeling proteins = cohesions, condensin, topoisomerase II
20
Q
Acentric fragment:
A
Absence of a centromere = unable to attach to spindle = lost chromosome = lost genetic information
21
Q
Dicentric/polycentric chromosome:
A
A single chromosome with 2 or more centromeres (commonly due to to fusion of two chromosomes)
Only one centromere is functional (centromere that most
efficiently assembles the kinetochore)
Allows normal cell division
22
Q
Features of the telomere:
A
- G-quartets/G-quadruplex
- T-Loop/D-Loop Formation
- Binding of the Shelterin Complex
- Telomerase Activity
23
Q
G-quartets/G-quadruplex function
A
Unknown!
Transient DNA structure
24
Q
What regulates telomere length?
A
Binding of shelterin complex and telomerase activity
25
Hierarchy of DNA Organization:
Ensures that the DNA is packaged in the most compact state.
26
RNA polymerase I synthesizes
5.8S, 18S, and 28S rRNA in the nucleolus.
27
RNA polymerase III synthesizes
tRNA, 5S rRNA genes, small
| RNAs in the nucleoplasm.
28
RNA polymerase II synthesizes
hnRNA; all RNA with the exception of those transcribed by RNA polymerase I & III
29
TFIIH
- Recruited by TFIIE
- Helicase activity
- Rapidly repairs damaged DNA to avoid RNAP II stalls.
- Phosphorylates the C-terminal domain (CTD) of RNAP II (functions in elongation, 5’ capping)
30
Epigenetics
Inheritable changes in a cell’s phenotype
| Independent of changes to the DNA sequence or genotype
31
Mechanism associated with Epigenetics:
1. Histone Post-translational Modifications
2. DNA Methylation
3. Regulatory RNA (non-coding RNA)
32
Histone acetylation
Activation of gene expression
33
Position-effect variegation (PEV)
Propagation of the inactive heterochromatin structure is
dictated by:
1. Availability of silencing proteins
2. Activation of promoters
3. Presence of insulator/boundary sequence.
Example: Drosophila eye color
34
Interrupted gene:
A gene in which the coding sequence is not continuous
| Therefore expressed via a precursor RNA
35
Prokaryotes have ___________ genes.
Prokaryotes have uninterrupted genes - DNA and mRNA sequences are the same.
36
Three classes of Introns:
1. Nuclear RNA
2. Group II
3. Group I
37
Similarity among all three classes of intron
All require two-step trans-esterification reactions!
38
Autosplicing introns
Group I, Group II
| NO ATP or GTP HYDROLYSIS for energy
39
Spliceosomes required for
Nuclear introns
| NEED ENERGY
40
Alternative Splicing:
A SINGLE GENE gives rise to more than one mRNA sequence due to changes in the splicing pattern of the
pre-mRNA
Contributes to the structural and functional
diversity of the gene product (either mRNA or protein)
41
E-complex
Commitment complex
Requires U1 at 5’ splice site
42
A-complex
Pre-spliceosome = ATP-dependent
U2-snRNP binding to the branch site
43
B1-complex
Spliceosome
Contains all splicing components
Formed with the binding of U5 and U4/U6-snRNPs
44
B2-complex
Release of U1 and U4
| Allows association of U6 with 5’ splice site and U2
45
C1- and C2-complexes
Transesterification and lariat structure
46
tRNA splicing:
ENDONUCLEASE cleaves the at 2 sites to dissociate
the intron
ATP-dependent LIGATION of the 2 exons
Forms a mature tRNA
47
Autosplicing of Group I introns:
Requires only guanosine and 2 metal ions.
GTP NOT hydrolyzed; no hydrolysis reaction occurs during self-splicing.
• Two-step transesterification
1. 3’OH of G nucleotide attacks phosphate at 5’-end of intron
2. 3’OH at 5’-exon attacks phosphate at second exon
• No lariat structure; the released intron engages in additional reactions.
48
Core regions of Group I introns
The cores of regions P3, P4, P6, and P7 form the minimal region required for catalytic activity
49
What mediates enzymatic reaction in a protein?
Ionic and hydrogen bonds mediate enzymatic reaction
50
What mediates enzymatic reaction in a ribozyme?
Base pairing with RNA substrate mediate enzymatic reaction
51
High affinity for substrate of ribozyme
= Low KM
52
Slow catalytic rate of ribozyme
= Low Kcat (turnover)
53
Protein Splicing
Autocatalytic process that excises an intein (analogous to RNA intron) and forms a peptide bond between the two adjacent exteins (analogous to RNA exons).
54
Does protein auto-splicing require energy?
Similar to RNA auto-splicing, protein auto-splicing does not require energy.
55
How do proteins mediate auto-splicing?
Similar to RNA, proteins mediate autosplicing via conformational changes through bond rearrangement.
Like mobile RNA intron, some protein inteins code for a homing endonuclease.
56
mRNA induced error
Wrong base
= MOST DETRIMENTAL
Not common
57
Ribosome induced error
Wrong aa-tRNA
= MOST COMMON
Not very detrimental
58
Termination codons are recognized not by charged-tRNAs but instead by
Protein release factors (RF).
59
Prokaryotes have ______ types of class 1 release factors...
Prokaryotes have two types of class 1 release factors (RF1 and RF2) and eukaryotes have one class 1 release factor (eRF1).
60
Both prokaryotes and eukaryotes require ________ for release...
Both prokaryotes and eukaryotes require the GTP-binding protein class 2 release factors (RF3 or eRF3).
61
The wobble hypothesis
Conventional base pairing between codon/anticodon occurs at the first 2 codons, but unconventional base pairing at the 3rd codon.
The unconventional base pairing occurs because ribosomal A site allows greater flexibility at the first anti-codon position.
U-G is a common base pair.
First anticodon may also be modified base.
62
Aminoacyl-tRNA synthetases are a family of enzymes that charge tRNA with an amino acid in a two-stage reaction:
1. ATP hydrolysis to form aminoacyladenylate.
| 2. A.A. transfer to tRNA and release AMP.
63
Each tRNA synthetase aminoacylates all the tRNAs...
In an isoaccepting group with the SAME amino acid.
64
Recognition of tRNA by tRNA synthetases is based on...
A particular set of nucleotides that often are concentrated in the acceptor stem and anticodon loop regions of the
molecule.
65
2/3 of RNA Poly II transcripts are cryptic unstable transcripts (CUTs)
- a subset of noncoding RNA (ncRNA)
- extremely unstable due to rapid degradation
- 3’ polyadenylation via TRAMP
- TRAMP recruitment of exosome responsible for rapid CUT
decay
66
Digestion of poly(A) tail to short oligoA tail
Triggers mRNA decay
67
5'-3' decay
Decapping by Dcp
Xrn1 eats fragments
5’ to 3’ exonuclease digestion by Lsm1-7
68
3'-5' decay
Exosome-mediated 3’ to 5’ digestion
69
If cell division
A new round of replication must be initiated before
the previous replication round is completed
Daughter cell is produced with replication already occurring with the presence of multi-forked chromosomes.
70
ftsZ mutations:
Multinucleated, long filaments form when septum formation is inhibited but replication is unaffected.
71
ftsZ overexpression:
No chromosome, small cells (called minicells) form when septum formation is either too rapid or not mid-cell.
Minicells different from anucleated mutants
72
Most of eukaryotic cells are in the...
Most of eukaryotic cells are not growing or in the G0 stage.
| Exception: stem cells, embryonic cells, and cancer cells.
73
Replication and cell division is controlled via:
Signal transduction pathway - process by which a stimulus or cellular state is sensed by and transmitted to pathways
within the cell.
Example: From the EGFR Signal Transduction Pathway.
74
Generating and Maintaining ssDNA is important for replication.
Requires two functions:
1. A helicase to separate/unwind the strands of DNA using energy provided by hydrolysis of ATP.
2. A single-stranded binding protein (SSB) is required to
maintain the separated strands.
75
A single-stranded binding protein (SSB) is required to:
Maintain the separated strands of DNA during replication.
76
There are several methods for providing the free 3’-OH
| end that DNA polymerases require to initiate DNA synthesis:
- RNA primer
- a nick in DNA
- a priming protein
77
RNA primer used:
- One time in leading strand
| - Multiple times in lagging strand
78
Primase
SPECIAL RNAP
Synthesizes RNA chain that provides priming end
Different from other RNAP!
79
Most 'important' to initiate DNA synthesis is
NICK in the DNA
80
DnaB
The helicase DnaB is responsible for interacting with the primase DnaG to initiate each Okazaki fragment
DnaB contacts the τ subunits of the clamp loader to connect the helicase-primase complex and the catalytic core.
81
Synthesis of the leading strand creates
A loop of ssDNA on the lagging strand
Enzyme complex pulls the lagging strand through the clamp while synthesizing the new strand
"Opposite direction" from leading strand - but still 5' to 3' synthesis
82
Bivalent
Synapse or chromosome pairing of all 4 chromatids during meiosis; this structure is called a bivalent.
Recombination initiated and strands exchanged.
83
Synaptonemal complex function
Stabilizes the synapse
| Could present an obstacle to recombination
84
Synaptonemal complex structure
Could present an obstacle to recombination.
• Loops of chromatin project from the axis.
• Cohesions connect sister chromatids so that they segregate properly at mitosis or meiosis. Form the lateral elements.
• Zip proteins transverse and link together to form the synaptonemal complex central element.
85
All recombination repair systems
REQUIRE LIGASE!
86
Direct Repair
Reversal/removal of the damage.
| LEAST common
87
Excision Repair
One strand of DNA is directly excised and then replaced
| by resynthesis using the complementary strand as template.
88
Mismatch Repair
Corrects recently inserted bases that do not pair
properly.
MOST common
89
Recombination Repair
Fills in a gap in one strand of duplex DNA by retrieving a homologous strand from another complex.
BEST to repair DSB
90
Non-homologous end-joining
Rejoins DSB
91
What happens to a replication fork when it encounters a damaged site or nick in DNA?
Stalls, then:
1) May reverse by pairing between the two newly synthesized strands
2) May restart after repairing the damage and use a helicase to move the fork forward
3) May initiate translesion/excision repair or recombination repair (also called “post-replication repair”)
92
Activator, when introduced, can...
Control kernel color in maize.
93
Activator (Ac) element
Autonomous transposable element in maize.
94
Dissociation (Ds) element
Non autonomous transposable element in maize, requires Ac.
95
Transcription generates ________ DNA ahead of
| RNA polymerase.
More tightly wound (positively supercoiled)
96
DNA behind RNAP becomes _________ during transcription.
Less tightly wound (negatively coiled)
97
Gyrase
Introduces negative supercoils (rectifies in front of RNAP)
98
Topoisomerase
Removes negative supercoils (rectifies behind RNAP)
99
Negative inducible
Active repressor + inducer => Inactive repressor
100
Negative repressible
Inactive repressor + corepressor => Active repressor
101
Positive inducible
Inactive activator + inducer => Active activator
102
Positive repressible
Active activator + corepressor => Inactive repressor
103
Polycistronic mRNA
mRNA that includes coding regions representing more than one gene
104
The cluster of the lac operon containing the 3 lac structural
genes
lacZ, lacY, and lacA.
105
lac repressor
```
Maintains the lac operon in the inactive condition by binding to the operator.
The repressor has 3 binding sites:
1) Operator DNA binding site
2) Inducer site
3) Multimerization site
```
106
Inducer for the lac repressor (negative inducible)
By-product of β-galactosidase, allolactose
107
cAMP is controlled by:
Level of glucose in the cell
Low glucose = High cAMP
High glucose = Low cAMP.
Glucose high = reducing the level of cyclic AMP = high glucose inhibits the transcription of operon
108
The trp operon is
The trp operon is negatively controlled by the level of its product (negative repressible), the amino acid tryptophan (autoregulation).
109
The amino acid tryptophan...
Activates an inactive repressor encoded by trpR gene.
110
The trp repressor recognizes operators at...
Three dispersed gene loci.
111
Lytic and lysogenic pathways both require...
Expression of the immediate early and delayed early genes.
112
Lytic development follows if...
The late genes are expressed.
| The lytic cycle requires the delayed early gene Q.
113
Lysogeny ensues if...
cI gene is expressed.
| Lysogeny requires the delayed early genes cII-cIII.
114
Lambda has 2 immediate early genes:
1. N gene codes for an antitermination factor.
2. Cro gene codes for a repressor that prevents the
expression of the cI gene.
115
The critical stage in deciding between lysogeny and lysis is...
WHEN delayed early genes are being expressed.
116
If cII causes sufficient synthesis of repressor...
Lysogeny will result because repressor occupies the
operators.
(Otherwise Cro occupies the operators, resulting in a lytic
cycle.)
117
Class II TE
= DNA elements
• Class I à Retroelements or RNA intermediates.
• LTR retrotransposon – A transposon that
mobilizes via an RNA form
- Similarity with a retrovirus à a virus with RNA
as its genetic material.
- like retrovirus, it expresses long-terminal repeats
(LTR) which are repetitive sequences analogous
to that of the provirus for integration into the
genome.
- like retrovirus, it’s RNA codes for and is subject
to an active reverse transcriptase.
- unlike retrovirus, it does not have a infective,
viral form.
• non-LTR retrotransposon (retroposon) – A
transposon that mobilizes via an RNA
intermediate, similar to an LTR retrotransposon,
but that lacks LTRs and uses a distinct
transposition mechanism.
118
Class I TE
Retroelements of RNA intermediates
119
LTR retrotransposon
A transposon that mobilizes via an RNA form
Similar to a RETROVIRUS!
Like retrovirus, expresses long-terminal repeats (LTR) = repetitive sequences analogous to a provirus = for integration into the genome.
RNA codes for and is subject to an active reverse transcriptase.
120
non-LTR retrotransposon (retroposon)
Mobilizes via an RNA intermediate
Lacks LTRs
Uses a distinct transposition mechanism
121
Transduction
Viral vector to release DNA into target cells
122
Transfection
Liposome bound, microinjection, nanospheres to release DNA into target cells
123
Sanger method
Chain termination sequencing
Uses dideoxynucleotides to terminate DNA synthesis at
particular nucleotides.
ddNTP = modified nucleotides that can be incorporated into a growing DNA strand, but lack the 3’-hydroxyl group needed to attach the next nucleotide.
124
Sequencing by synthesis (SS-sequencing)
Relies on the detection of each nucleotide as it is added to a growing strand.
Example #1: Tethered primer and fluorescent-dNTP
Example #2: Pyrosequencing is a process to detect
the release of the pyrophosphate (diphosphate) with the addition of the nucleotide to a growing strand; detection via
bioluminescence.
125
Real-time PCR
Real-time, or quantitative, PCR detects the products
| of PCR amplification during their synthesis, and is more sensitive and quantitative than conventional PCR.
126
Ct
Threshold cycle = when the amplicon/product can be detected
127
MCS
MCS (multiple cloning site) = A sequence of DNA containing a series of tandem restriction endonuclease sites, used in
cloning vectors for creating recombinant molecules.
Insert between two MCS sites.
128
Staggered cuts
Requires SAME restriction enzyme for Vector and Insert.
129
Blunt cuts
Do NOT require the same restriction enzyme for Vector and Insert
